DNA replication is central to the life cycle of every living organism. Although considerable progress has been made in our understanding of how this process is regulated in simple organisms, regulatory mechanisms in higher eukaryotes remain largely unknown. By introducing Chinese hamster ovary (CHO) cell nuclei into Xenopus egg extracts, the applicant has produced the first cell-free system that will initiate DNA replication preferentially at a physiologically utilized origin of replication downstream of the dihydrofolate reductase (DHFR) gene. Recognition of this origin requires some component of the CHO nucleus that is assembled at a discrete point during G1-phase (Origin Decision Point, ODP), after replication licensing and prior to restriction point control. After each mitosis, the cell must re-assemble a highly organized and functionally compartmentalized nucleus. In particular, replication takes place at fixed sites within the nucleus that consist of multiple coordinately regulated replicons joined to large (approximately 0.1 microns) multiprotein complexes. Our working hypothesis predicts that ODP represents the joining of replication origins to this multiprotein complex. To address this hypothesis, we will pulse label CHO cells with BrdU within the first 10 minutes of S- phase and chase these cells through to the following mitosis. Synchronized populations of CHO cells, containing BrdU-tagged origin- proximal sequences, will then be collected at various times during G1- phase and analyzed by fluorescence microscopy for the time at which these sequences 1) re-establish their early S-phase pattern of foci, 2) become attached to a fixed nuclear substratum, 3) become functionally recognizable as the sites at which to begin DNA synthesis when nuclei from these cells are introduced into Xenopus egg extracts, and 4) first co-localize with antibodies directed against essential replication initiation factors. Parallel experiments will monitor the spatial position and attachment to the matrix of specific origin-containing and non-origin probes that decorate the DHFR replicon using Fluorescence In Situ Hybridization to CHO cells synchronized in different stages of G1- phase. Having established the sequence of these events, we will disrupt the assembly of the nucleus through 1) the controlled overexpression of dominant negative nuclear lamina proteins that have been shown to interfere with the initiation of replication and 2) treatment of cells with inhibitors of Topoisomerase II, one of which has been shown to inhibit the selection of origins at the ODP. We will then determine which steps in the assembly of functional replication origins are interrupted by these disruptions in nuclear structure.